Leishmania synthesize abundant phosphoglycan-containing molecules made up of Gal-Man-PO4 repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). LPG, the major cell surface molecule of Leishmania promastigotes, is known to mediate parasite attachment to the vector midgut, which prevents the loss of infection during excretion of the bloodmeal remnants. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis was tested using L. major mutants specifically deficient in either total phosphoglycans (LPG2KO) or LPG alone (LPG1KO), along with their respective gene add back tranfectants. Our results showed that 48-72 hours after the infective feed, all parasites developed well except the LPG2KO, which showed reduced survival and growth of promastigotes. Following passage of the digested bloodmeal (7-21 days), neither the LPG1KO nor LPG2KO parasites were able to survive in P. duboscqi, although the LPG1KO continued to survive in L. longipalpis. Inhibitors of trypsin and alkaline protease activities promoted the early midgut survival of the LPG2KO. Phosphoglycosylated PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of LPG2KO promastigotes exposed to midgut lysates prepared from bloodfed flies. [unreadable] A major effort has been directed toward quantification of the natural transmission dose of Leishmania to mammalian host during the bite of an infected sand fly, which is currently unknown for any natural sand fly vector. The quantitative data are needed to define the dose of parasites that should be used when designing experimental needle infections intended to mimic natural transmission, and revealing natural dose ranges that may influence clinical outcome. We developed a real-time PCR-based method to determine the number of Leishmania major parasites inoculated into the ears of living mice during feeding by a single infected infected fly (Phlebotomus dubosqui). Most of the infected mice were inoculated with a low dose of fewer than 260 Leishmania parasites. However, one in four received a higher dose of up to 100,000. To test the impact of the size of the innoculum on disease outcome, we compared high (5000) and low (100) dose intradermal needle infections in the ears of C57BL/6 mice. To mimic natural transmission, we used sand fly derived metacyclic forms of L. major and pre-exposed the injection site to the bites of un-infected flies. As expected, pathology was significantly worse in the mice receinving the higher dose, but unexpectedly, the number of persisting parasites following healing was higher in the low dose challenged mice. The results indicate that transmitted dose influences the severity of disease and infection reservoir potential. [unreadable] Transmission by sand fly bite is being used for the first time to evaluate the efficacy of experimental vaccines against leishmaniasis. A variety of non-living, recombinant protein, and DNA based vaccines have been shown to confer protection in animal models. Remarkably, no experimental vaccine has been evaluated by sand fly challenge in a controlled, laboratory setting. We have recently refined our ability to efficiently transmit cutaneous leishmanisis to mice using infected sand flies, and have produced a clear set of data to indicate that the non-living vaccines that confer powerful protection against needle challenge are ineffective against sand fly challenge. As sand fly bites are associated with a massive recruitment of neutrophils to the site of parasite deposition in the skin, the accumulating evidence that neutrophils are the initial cellular target of metacyclic promastigotes and are employed as means of silent parasite entry into macrophages, may explain the compromised expression of immunity in the vaccinated mice exposed to infected flies. Employing a red fluorescent protein (RFP) expressing strain of L. major we investigated the cellular target of metacyclic promastigotes at acute time points post-infection in the ear dermis. We report that 75-80% of parasites are contained within neutrophils at 20 hours post-infection. In contrast, by 48 hours post-infection 50% of parasites are found within macrophages. Infected neutrophils are not apoptotic and contain viable parasites that can be propagated in culture. Sorted infected neutrophils established infection upon injection into nave mice similar to that of cultured parasites. Employing two-photon microscopy we were able to visualize parasite dependent neutrophil recruitment and subsequent parasite phagocytosis by neutrophils. These findings strongly suggest that L. major preferentially recruits and infects neutrophils during acute infection and these infected neutrophils fail to kill intracellular parasites, thereby supporting the Trojan Horse Model of acute Leishmania infection in which L. major employs neutrophils as a means by which to gain silent entry into macrophages.